Rhinovirus Wheezing Illness and Genetic Risk of Childhood-Onset Asthma

The
n e w e ng l a n d j o u r na l
of
m e dic i n e
original article
Rhinovirus Wheezing Illness and Genetic

Risk of Childhood-Onset Asthma
Minal alkan, B.S., Yury A. Bochkov, Ph.D., Eskil Kreiner-Mller, M.D.,
Klaus Bnnelykke, M.D., Michelle M. Stein, B.S., Gaixin Du, M.S.,
Hans Bisgaard, D.M.Sci., M.D., Daniel J. Jackson, M.D., James E. Gern, M.D.,
Robert F. Lemanske, Jr., M.D., Dan L. Nicolae, Ph.D., and Carole Ober, Ph.D.
A BS T R AC T
BACKGROUND
From the Departments of Human Genetics (M.., M.M.S., G.D., D.L.N., C.O.),
Medicine (D.L.N.), Statistics (D.L.N.), and
Obstetrics and Gynecology (C.O.), University of Chicago, Chicago; the Departments of Pediatrics (Y.A.B., D.J.J., J.E.G.,
R.F.L.) and Medicine (R.F.L.), University of
WisconsinMadison, Madison; and Copenhagen Prospective Studies on Asthma
in Childhood, Faculty of Health Sciences,
University of Copenhagen; the Danish
Pediatric Asthma Center; and Copenhagen
University Hospital, Gentofte all in
Copenhagen (E.K.-M., K.B., H.B.). Address
reprint requests to Ms. alkan or Dr.
Ober at the Department of Human Genetics, University of Chicago, 920 E. 58th St.,
Chicago, IL 60637, or at caliskan@uchicago
.edu or c-ober@genetics.uchicago.edu.
Both genetic variation at the 17q21 locus and virus-induced respiratory wheezing
illnesses are associated with the development of asthma. Our aim was to determine
the effects of these two factors on the risk of asthma in the Childhood Origins of
Asthma (COAST) and the Copenhagen Prospective Study on Asthma in Childhood
(COPSAC) birth cohorts.
This article was published on March 27,

2013, at NEJM.org.
The 17q21 variants were associated with HRV wheezing illnesses in early life, but not
with RSV wheezing illnesses. The associations of 17q21 variants with asthma were
restricted to children who had had HRV wheezing illnesses, resulting in a significant interaction effect with respect to the risk of asthma. Moreover, the expression
levels of ORMDL3 and of GSDMB were significantly increased in HRV-stimulated
PBMCs, as compared with unstimulated PBMCs. The expression of these genes was
associated with 17q21 variants in both conditions, although the increase with exposure to HRV was not genotype-specific.
N Engl J Med 2013;368:1398-407.

DOI: 10.1056/NEJMoa1211592
Copyright 2013 Massachusetts Medical Society.
METHODS
We tested genotypes at the 17q21 locus for associations with asthma and with human
rhinovirus (HRV) and respiratory syncytial virus (RSV) wheezing illnesses and tested
for interactions between 17q21 genotypes and HRV and RSV wheezing illnesses with
respect to the risk of asthma. Finally, we examined genotype-specific expression of
17q21 genes in unstimulated and HRV-stimulated peripheral-blood mononuclear
cells (PBMCs).
RESULTS
CONCLUSIONS
Variants at the 17q21 locus were associated with asthma in children who had had
HRV wheezing illnesses and with expression of two genes at this locus. The expression levels of both genes increased in response to HRV stimulation, although the
relative increase was not associated with the 17q21 genotypes. (Funded by the National Institutes of Health.)
1398
n engl j med 368;15 nejm.org april 11, 2013
The New England Journal of Medicine

Downloaded from nejm.org on December 1, 2014. For personal use only. No other uses without permission.
Copyright 2013 Massachusetts Medical Society. All rights reserved.
Wheezing and Genetic Risk of Asthma
he first genomewide association

study of childhood-onset asthma revealed
a susceptibility locus on chromosome
17q21.1 The association of this locus with asthma has since been replicated in both genomewide and candidate-gene association studies,2,3
and the locus represents one of the most con
sistently associated genetic risk factors for childhood asthma. Variation at the 17q21 locus is
associated primarily with childhood-onset asthma,4-6 but not with atopy,4,7,8 and the effects are
larger among children who had been exposed to
environmental tobacco smoke in early life4,9-11
and in children with reported respiratory infections in infancy.10 The disease-associated variants at this locus are associated with expression
levels of two 17q21 genes, GSDMB and ORMDL3,
in white cells,5 lymphoblastoid cell lines,12 and
CD4+ T cells.13
The onset and progression of asthma result
from a complex interplay between genetic background and environmental exposures, particularly
in early development. Among the many environmental factors that influence the risk of asthma,14
respiratory infections with viruses15,16 and bacteria17 are the most common triggers of asthma
exacerbations in children. Approximately 80% of
asthma exacerbations are attributed to respiratory
viral infections, with human rhinovirus (HRV)
accounting for nearly two thirds of these cases.16
Moreover, infants who have HRV infections with
wheezing are at a significantly increased risk for
subsequent asthma.18-20 However, exposure to HRV
does not lead to wheezing illness in all children,
nor does wheezing illness result in asthma in all
cases, suggesting that the host genotype also plays
a role.
We sought to further elucidate the effect of
the genotype at the 17q21 asthma locus and respiratory viral illnesses in early life on the risk
of childhood-onset asthma. We hypothesized
that these common genetic and environmental
risk factors account for a substantial proportion
of the overall risk of childhood asthma, in either
an additive or an interactive manner. We also
used an in vitro model in peripheral-blood
mononuclear cells (PBMCs) to identify HRVresponsive genes at the 17q21 locus and to test
a potential mechanism underlying the inter
action.
Me thods
Study Participants
We included data from two cohorts of children

the Childhood Origins of Asthma (COAST)
birth cohort and the Copenhagen Prospective
Study on Asthma in Childhood (COPSAC) birth
cohort and a group of adult volunteers. Children who participated in the COAST birth cohort
provided oral assent when possible, and their parents provided written informed consent; oral and
written informed consent were provided by both
parents of each child who participated in the
COPSAC birth cohort. Adult participants provided
written informed consent. The study protocols for
the three cohorts were approved by the institutional review board at the University of Wisconsin,
the University of Copenhagen, and the University
of Chicago, respectively.
For the COAST study, 289 newborns were enrolled in Madison, Wisconsin, between November
1998 and May 2000, as described previously.21 All
the children had at least one parent with respiratory allergies, a history of physician-diagnosed
asthma, or both. The parents of 214 of the newborns who were of European ancestry gave consent for their child to participate in genetic studies, and data from these children are included in
the current analyses. A total of 200 of these children were evaluated for asthma beginning at
6 years of age. The demographic characteristics
of the COAST cohort are provided in Table S1 in
the Supplementary Appendix, available with the
full text of this article at NEJM.org. The diagnostic criteria for asthma and descriptions of allergic
sensitization and phenotypes of viral wheezing
illnesses in the COAST cohort are provided in the
Methods section of the Supplementary Appendix.
For COPSAC, 411 Danish children (all of European ancestry) born to mothers with a history
of physician-diagnosed asthma were enrolled between August 1998 and December 2001 in Copenhagen.22 A total of 297 of these children with
complete follow-up data from the first 3 years of
life and with information on asthma status by
7 years of age were included in this study. The
demographic characteristics of the COPSAC cohort are provided in Table S1 in the Supplementary Appendix. The diagnostic criteria for asthma
and descriptions of phenotypes of viral wheezing

1399
The
illnesses in the COPSAC cohort are provided in the

Methods section of the Supplementary Appendix.
In addition, we recruited 100 unrelated adult
volunteers (49 men and 51 women; age range, 19
to 60 years) in Chicago between July and November 2011 in order to study the genotype-specific
effects of HRV stimulation on gene-expression
patterns in PBMCs. We recruited volunteers by
posting flyers (which were approved by the institutional review board) around the University of
Chicago campus. Each participant responded to
a short medical questionnaire on the day the
blood samples were obtained; 23 of the participants reported a previous diagnosis, by a physician, of asthma. According to self-reported ancestry, 84 of the participants were of European
descent, 8 were of Asian descent, 3 were Hispanic, and 4 had mixed ancestry; 1 participant
did not report ancestry.
Genotyping of 17q 21 Variants
Five asthma-associated 17q21 single-nucleotide

polymorphisms (SNPs) rs9303277, rs11557467,
rs12936231, rs2290400, and rs7216389 (Fig. S1
in the Supplementary Appendix) were genotyped in the COAST cohort. Genotyping was performed at the National Heart, Lung, and Blood
Institute Resequencing and Genotyping (RS&G)
Service at Johns Hopkins University, with the
use of the Custom GoldenGate Genotyping assay
(Illumina). Success rates of genotyping ranged
from 98.6 to 100%. SNP rs7216389 was selected
as a surrogate for the five SNPs that are in near
perfect linkage disequilibrium (Fig. S1 in the Supplementary Appendix). This SNP was genotyped
in the COPSAC and adult participants with the use
of TaqMan assays (Applied Biosystems).
of
m e dic i n e
covariates. In addition, the number of wheezing

episodes in which another (i.e., non-HRV or nonRSV) virus was present was included as a covariate to ensure that the observed effects were specific to the virus being tested. Details of the
statistical methods are provided in the Supplementary Appendix.
HRV16 Stimulation of PBMC s and DNA
and RNA Extraction
A blood sample (20 ml) was obtained from each

adult participant. PBMCs were isolated from wholeblood samples with the use of a Ficoll-Paque
separation protocol. A total of 4106 PBMCs from
each participant were treated with medium alone
for 24 hours, and 4106 PBMCs were treated with
medium containing HRV16 for 24 hours. The
multiplicity of infection was 10 plaque-forming
units per cell. The remaining cells from each participant were used for DNA extraction. DNA was
extracted on the day the blood samples were obtained, with the use of the QIAamp DNA Blood
Mini Kit (Qiagen). Total RNA was extracted after
a 24-hour incubation period, with the use of the
RNeasy Plus Mini Kit (Qiagen). Details of the experimental procedures are provided in the Methods section of the Supplementary Appendix.
Complementary DNA Synthesis
and Quantitative PCR Assay
Complementary DNA (cDNA) was synthesized with

the use of the SuperScript III First-Strand Synthesis System and oligo-dT primers (Invitrogen). Primers for five 17q21 genes (IKZF3, ZPBP2, GSDMB,
ORMDL3, and GSDMA) and a housekeeping gene
(POLR2C) were designed with the use of IDT SciTools (Integrated DNA Technologies) (Table S2
in the Supplementary Appendix). A quantitative
Genetic Association and Interaction Studies polymerase-chain-reaction (qPCR) assay was perEach SNP was tested for an association with formed with the use of Platinum SYBR Green
asthma and with phenotypes of viral wheezing qPCR SuperMix-UDG (Invitrogen) and the 7900HT
illness, with the use of a logistic-regression model Fast Real-Time PCR system (Applied Biosystems).
if the outcome variable was binary or a linearregression model if the outcome variable was Analysis of Gene-Expression Data
continuous. The model included the phenotype Among five genes at the 17q21 locus, IKZF3, GSDMB,
of interest as the outcome variable and genotype and ORMDL3 were successfully amplified in unas an explanatory variable. To test for interac- stimulated and HRV-stimulated PBMCs obtained
tions between 17q21 SNP genotypes and viral from 100 participants. Each qPCR reaction was
(HRV or RSV) wheezing illnesses, viral wheezing run in duplicate, and only samples with a coefillness and a term for the interaction of viral ficient of variation of less than 0.02 were includwheezing illness with genotype were included as ed in the analyses. After the exclusions, there were
1400

Table 1. Associations of 17q21 Single-Nucleotide Polymorphisms (SNPs) with Asthma, Allergic Sensitization, and Viral Wheezing Illness
Phenotypes in the Childhood Origins of Asthma (COAST) Cohort.*
SNP
Gene
Location
Minor
Allele
Minor-Allele
Frequency
P Value
HRV
Allergic
Wheezing
Asthma Sensitization
Illness
No. of HRV
Wheezing
Illnesses
RSV
Wheezing
Illness
No. of RSV
Wheezing
Illnesses
rs9303277
IKZF3
Intron
0.488
0.03
0.86
0.01
<0.001
0.25
0.51
rs11557467
ZPBP2
Intron
0.498
0.05
0.98
0.02
<0.001
0.30
0.63
rs12936231
ZPBP2
Exon
0.493
0.07
0.95
0.02
<0.001
0.22
0.57
rs2290400
GSDMB
Intron
0.500
0.08
0.90
0.02
<0.001
0.17
0.47
rs7216389
GSDMB
Intron
0.500
0.04
0.90
0.01
<0.001
0.22
0.54
* HRV denotes human rhinovirus, and RSV respiratory syncytial virus.

Allergic sensitization was assessed with the use of an in vitro IgE test. The results were similar when allergic sensitization was assessed
with the use of a skin-prick test (all P>0.50).
This was the minor allele in the COAST cohort.
The minor allele in Centre dEtude du Polymorphisme Humain from Utah (CEU) HapMap samples is shown for this SNP.
96 paired samples of unstimulated and HRVstimulated PBMCs for expression studies of IKZF3,
97 paired samples for expression studies of GSDMB,
and 97 paired samples for expression studies of
ORMDL3. Additional details of the qPCR data analysis are provided in the Methods section of the
Supplementary Appendix.
We used a paired t-test to assess differences in
the expression of each 17q21 gene (IKZF3, GSDMB,
and ORMDL3) between unstimulated and HRVstimulated cells. Associations between 17q21 SNPs
and gene expression were tested under an additive
genetic model.
R e sult s
Associations in the COAST Cohort
All the SNPs showed modest associations with

childhood-onset asthma in 200 children in the
COAST cohort (P0.08) (Table 1 and Fig. 1A). The
SNPs that were tested were in nearly perfect linkage disequilibrium (Fig. S1 in the Supplementary
Appendix) and had nearly identical minor-allele
frequencies (0.49 to 0.50) (Table 1), making it impossible to assign the association signal to any
particular SNP at the locus. None of the SNPs were
associated with allergic sensitization (P0.86)
(Table 1), a finding that was consistent with the
results of previous studies.4,7,8 The 17q21 SNPs
also had significant associations with HRV wheezing illness (P0.02) (Table 1) and with the number of HRV wheezing illnesses (P<0.001) (Table 1
and Fig. 1B) in the first 3 years of life. None of

the SNPs were associated with either the risk of
RSV wheezing illnesses (P0.17) or the number
of RSV wheezing illnesses (P0.47) (Table 1), indicating the specificity of the association with
HRV wheezing illness.
Interactions between 17q21 Variants
and Viral Wheezing Illnesses
We next examined interactions between the 17q21

genotype and HRV and RSV wheezing illnesses
with respect to the risk of asthma in the COAST
cohort. We observed significant interactions between 17q21 genotypes and HRV wheezing illness with respect to the subsequent risk of asthma (P0.01 for interaction) (Fig. 1C, and Table S3
in the Supplementary Appendix). The association
between genotypes and asthma was present in
children with HRV wheezing illness (odds ratio
for the TT genotype at rs7216389, 26.1; 95% confidence interval [CI], 5.1 to 133.0) (Fig. 1D, and
Table S3 in the Supplementary Appendix) but not
in children without HRV wheezing illness (odds
ratio for the TT genotype at rs7216389, 0.8; 95%
CI, 0.2 to 2.4) (Fig. 1E, and Table S3 in the Supplementary Appendix). The odds ratio for childhood asthma among children with the TT genotype by itself was 2.3 (95% CI, 1.0 to 5.2), and the
odds ratio for childhood asthma among children
with HRV wheezing illness by itself was 5.2 (95%
CI, 2.8 to 9.9). In contrast, no significant interaction was observed with RSV wheezing illness

1401
The
60
Asthma (%)
50
P=0.04
40
30
20
10
0
m e dic i n e
B Mean No. of HRV Wheezing Illnesses

No. of HRV Wheezing Illness
A Prevalence of Asthma
of
CC
(51)
CT
(100)
TT
(49)
2.5
P<0.001
2.0
1.5
1.0
0.5
0.0
CC
(52)
CT
(110)
rs7216389
rs7216389
C Prevalence of Asthma According to History of HRV
D Asthma Risk, Children with HRV Wheezing Illnesses
Wheezing Illnesses
Odds Ratio (log10)
100
P=0.004 for interaction
90
80
1 Wheezing illnesses
Asthma (%)
1
0
1
60
CC
(12)
CT
(33)
TT
(20)
rs7216389
50
40
E Asthma Risk, Children without HRV Wheezing Illnesses
30
No wheezing illnesses
10
CC
CT
TT
rs7216389
Odds Ratio (log10)
20
2
1
0
1
2
No. with Genotype

70
TT
(52)
12
39
33
67
20
29
CC
(39)
CT
(67)
TT
(29)
rs7216389
Figure 1. Effects of 17q21 Genotype on Asthma and Human Rhinovirus (HRV) Wheezing Illnesses in the Childhood
Origins of Asthma (COAST) Cohort.
Panel A shows the prevalence of asthma according to 17q21 genotype, and Panel B shows the mean number of HRV
wheezing illnesses in the first 3 years of life according to 17q21 genotype. Sample sizes for each genotype group are
shown in parentheses under the horizontal axis. T bars in Panel B indicate standard errors. Panel C shows the prevalence of asthma among the children according to the whether they had at least one HRV wheezing illness in the
first 3 years of life or no HRV wheezing illness. The dashed horizontal line shows the overall prevalence of asthma
among children in the COAST cohort. P=0.006 for the main effect of this single-nucleotide polymorphism (SNP)
among children who had at least one HRV wheezing illness in the first 3 years of life, and P=0.70 among children
who did not have an HRV wheezing illness; P=0.004 for the interaction between the rs7216389 SNP and HRV wheezing illness with respect to the development of asthma. Panel D shows the odds-ratio estimates for asthma risk associated with the three genotypes at rs7216389 among children who had at least one HRV wheezing illness in the first
3 years of life, and Panel E, the odds-ratio estimates among children who did not have any HRV wheezing illnesses in
the first 3 years of life. In Panels D and E, the odds ratios are shown relative to the reference group the children
with the CC genotype who did not have any HRV wheezing illnesses. The sample size of each genotype group is
shown in parentheses under the horizontal axis in both panels, and I bars indicate 95% confidence intervals.
(P0.30 for interaction) (Table S4 in the Supple- (17q21 genotype) and a common environmental
mentary Appendix). These data suggest an inter- risk factor (HRV wheezing illness) with respect to
action between a common genetic risk factor childhood-onset asthma.
1402

A Prevalence of Asthma According to History of HRV
B Asthma Risk, Children with HRV Wheezing Illnesses
Wheezing Illnesses
Odds Ratio (log10)
40
P=0.08 for interaction

Asthma (%)
30
2
1
0
1
2
CC
(12)
20
CT
(32)
TT
(29)
rs7216389
C Asthma Risk, Children without HRV Wheezing Illnesses
CC
CT
TT
rs7216389
No. with Genotype
12
59
32
109
29
56
Odds Ratio (log10)
10
2
1
0
1
2
CC
(59)
CT
(109)
TT
(56)
rs7216389
Figure 2. Effects of 17q21 Genotype on Asthma and HRV Wheezing Illnesses in the Copenhagen Prospective Study
on Asthma in Childhood (COPSAC) Cohort.
Panel A shows the prevalence of asthma among children in the COPSAC cohort, according to whether they had at
least one HRV wheezing illness in the first 3 years of life or no HRV wheezing illnesses. The dashed horizontal line
shows the overall prevalence of asthma among children in the COPSAC cohort. Panel B shows the odds-ratio estimates
for asthma risk associated with the three genotypes at rs7216389 among children who had at least one HRV wheezing illness in the first 3 years of life, and Panel C, the odds-ratio estimates for asthma risk associated with the three
rs7216389 genotypes among children who did not have any HRV wheezing illnesses in the first 3 years of life. In both
panels, the odds ratios were relative to the reference group children with the CC genotype who did not have any
HRV wheezing illnesses. In Panels B and C, the numbers in parentheses under the horizontal axis are the numbers
of participants with a given genotype. I bars in Panels B and C indicate 95% confidence intervals.
Replication Studies in the COPSAC Cohort
We next studied this interaction in the COPSAC

birth cohort22 and found that 17q21 genotypes
were associated with asthma in this cohort
(P=0.01 for the association with rs7216389)7 and
in the subgroup of 297 children included in this
study (P=0.02 for the association with rs7216389).
We stratified the children in the COPSAC cohort
according to the presence or absence of HRV wheezing illness in the first 3 years of life (Fig. 2A)
an approach similar to the one we used in the
COAST cohort.
We observed that the overall prevalence of
asthma was lower in the COPSAC cohort than in
the COAST cohort, a finding that we attribute to
two differences between the cohorts. First, the
rate of HRV wheezing illness, an important risk
factor for asthma, was lower in the COPSAC co-
hort than in the COAST cohort (24.6% vs. 31.3%).

Second, the criteria for the diagnosis of asthma
differed between the two cohorts. In COPSAC,
the diagnosis of asthma required a response to
a 3-month course of inhaled glucocorticoids followed by a relapse after treatment was stopped
(persistent asthma). In the COAST study, the diagnosis of asthma was based either on the documented use of rescue albuterol, intermittent use
of agents to control asthma, or short courses of
oral glucocorticoids only (intermittent asthma) or
on the daily use of medications to control asthma
(persistent asthma). Nonetheless, we found that
in the COPSAC cohort, the association between
17q21 genotypes and asthma was present in children who had had at least one HRV wheezing
illness in early life (odds ratio for the TT genotype at rs7216389, 3.9; 95% CI, 1.3 to 11.7) (Fig.

1403
The
A IKZF3
m e dic i n e
B GSDMB
C ORMDL3
P=0.95
P<0.001
P<0.001
HRV
Relative Expression
Relative Expression
Relative Expression
of
HRV
HRV
Figure 3. Expression of IKZF3, GSDMB, and ORMDL3 in Peripheral-Blood Mononuclear Cells (PBMCs) from the
Adult Participants.
Relative expression levels are shown in unstimulated (U) and HRV-stimulated (HRV) PBMCs. Expression of IKZF3 was
measured in 96 paired samples, and expression of GSDMB and ORMDL3 in 97 paired samples. Each circle corresponds
to an individual participant. The horizontal lines show the mean expression levels. As compared with the expression
of IKZF3, GSDMB, and ORMDL3 in unstimulated cells, the mean expression of these genes in HRV-stimulated cells
was increased by a factor of 1.01, 1.29, and 2.17, respectively.
2B, and Table S5A in the Supplementary Appendix) but not in children who had not had an HRV
wheezing illness (odds ratio for the TT genotype
at rs7216389, 0.7; 95% CI, 0.2 to 2.4; P=0.08 for
interaction) (Fig. 2C, and Table S5A in the Supplementary Appendix). The difference in the P values
for interaction between the COAST and COPSAC
birth cohorts is probably due to the smaller number of children with asthma in the COPSAC cohort. There was no interaction between 17q21
genotype and RSV wheezing illness in the COPSAC
cohort, a finding consistent with that in the
COAST cohort (P=0.29 for interaction) (Table S5B
in the Supplementary Appendix). A combined
analysis (meta-analysis) of the results in the COAST
and COPSAC samples, with the use of Fishers
test, yielded a P value of 0.003 for the interaction
between 17q21 genotypes and HRV wheezing
illness and a P value of 0.30 for the interaction
between 17q21 genotypes and RSV wheezing illness with respect to the risk of asthma.
Transcriptional Response to HRV Stimulation
We hypothesized that exposure to HRV might alter the expression levels of one or more of the
17q21 genes, possibly in a genotype-specific fashion. To test this hypothesis, we examined the expression patterns of the five 17q21 genes in unstimulated PBMCs and in HRV-stimulated PBMCs
1404
from 100 adults. Of the five genes at this locus,

only IKZF3, GSDMB, and ORMDL3 transcripts were
amplified in PBMCs in both stimulated and unstimulated conditions. As compared with the expression of IKZF3, GSDMB, and ORMDL3 in unstimulated cells, the expression of these genes in
HRV-stimulated cells was increased by a factor of
1.01, 1.29, and 2.17, respectively (P=0.95, P<0.001,
and P<0.001, respectively) (Fig. 3).
To examine genotype-specific effects on transcript levels of the three expressed genes in unstimulated cells and in HRV-stimulated cells, we
stratified participants according to genotype at
rs7216389. The rs7216389 genotype was associated with transcript levels of GSDMB and ORMDL3,
but not IKZF3, in both unstimulated and HRVstimulated cells (Fig. 4), a finding that was consistent with the results of previous studies.5,12,13
The relative increase in the expression of GSDMB
and ORMDL3 after exposure to HRV was not, however, associated with 17q21 genotype (Fig. S2 in
the Supplementary Appendix). These same trends
were observed when we limited our studies to participants of European ancestry. In addition, in genomewide gene-expression data from the same
100 participants, the 17q21 genotypes were not
significantly associated with expression levels of
any non-17q21 genes in either unstimulated or
HRV-stimulated cells (data not shown).

A IKZF3, rs7216389
P=0.11
Unstimulated
Relative Expression
Figure 4. Association between rs7216389 Genotype

and Expression of 17q21 Genes in Unstimulated
and HRV-Stimulated PBMCs.
Relative expression levels of IKZF3, GSDMB, and
ORMDL3 are shown according to 17q21 genotype in
unstimulated and HRV-stimulated PBMCs. The sample
size of each genotype group is shown in parentheses
under the horizontal axis. Each circle corresponds to
an individual participant. The horizontal lines show
the mean expression levels. The P values indicate the
significance of the association between the number of
T alleles and gene expression.
CC
(25)
CT
(41)
TT
(30)
CC
(25)
CT
(41)
TT
(30)
B GSDMB, rs7216389
Relative Expression
P<0.001
Unstimulated
P<0.001
HRV-stimulated
CC
(26)
CT
(41)
TT
(30)
CC
(26)
CT
(41)
TT
(30)
C ORMDL3, rs7216389
P=0.07
Unstimulated
Relative Expression
We have found a significant interaction, in both

the COAST and COPSAC birth cohorts, between
17q21 genotypes and HRV wheezing illness in
early life with respect to the development of
childhood-onset asthma, such that associations
between 17q21 genotypes and asthma are restricted to children with HRV wheezing illness in
early childhood. These findings are consistent
with those of a previous study that showed interactions between SNPs at the 17q21 locus and retrospective reports of respiratory illnesses in early
life (P=0.02 for interaction)10 and with reports of
more significant associations between 17q21 variants and asthma among children exposed to environmental tobacco smoke in early life than among
those without such exposure.4,9-11 Exposure to
environmental tobacco smoke is associated with
increased rates of early viral illnesses,23 and a
large body of literature links both exposure to
environmental tobacco smoke and early viral illnesses to an increased risk of asthma.18-20,23 Our
results implicate an interaction between HRV
wheezing illness in early childhood and 17q21
genotype. The odds ratio for childhood asthma
among children in the COAST cohort who were
TT homozygotes at rs7216389 and had had a
wheezing HRV illness was 26.1 (95% CI, 5.1 to
133.0), as compared with odds ratios for childhood asthma of 2.3 (95% CI, 1.0 to 5.2) with the
TT genotype by itself and 5.2 (95% CI, 2.8 to 9.9)
with HRV wheezing illness by itself; the effects
of each are not merely additive but are due to an
interaction.
There are some limitations of our study. First,
because HRV is the most common virus encountered, we cannot exclude the possibility that the
absence of an observed interaction with RSV is
Discussion
P=0.16
HRV-stimulated
P=0.03
HRV-stimulated
CC
(24)
CT
(42)
TT
(31)
CC
(24)
CT
(42)
TT
(31)
due to insufficient power. Second, we analyzed

viral exposure in children with wheezing illness,
and we cannot establish causality; it may be that
HRV wheezing illness is merely a marker of the

1405
The
underlying predisposition for asthma and is not

causal. Finally, we were unable in this study to
evaluate the role of concomitant bacteria, which
also triggers episodes of wheezing.17
Although the functions of the genes at the
17q21 locus are not well understood and the
mechanisms leading to asthma are unknown, it
has previously been suggested that ORMDL3 may
play a role in viral respiratory infections.24 The
ORMDL3 protein localizes to the endoplasmic
reticulum membrane, where it regulates endoplasmic reticulummediated calcium signaling
and the unfolded-protein response,25 probably
through the ATF6 pathway.26 The induction and
manipulation of unfolded-proteinresponse signaling are mechanisms through which viruses
protect host cells from death mediated by endoplasmic reticulum stress.27 The significant upregulation of ORMDL3 transcript in HRV-stimulated cells and the association between 17q21
variants and the number of HRV wheezing illnesses are consistent with this role and suggest
that overexpression of ORMDL3 may increase the
efficiency of the infection or viral replication in
respiratory epithelial cells, which are the site of
HRV infection and replication, and possibly reduce the ability of these cells to repair themselves after HRV infection. Little is known about
GSDMB, other than that it belongs to the cancerassociated gasdermin domaincontaining protein
family28,29 and might be involved in secretory
pathways28 and stem-cell proliferation in normal
of
m e dic i n e
epithelia.29 Further studies of the effects of the

17q21 genotype in response to HRV infection in
respiratory epithelial cells and in the presence of
concomitant bacteria could shed additional light
on the mechanism for the observed interaction.
Moreover, studies of the effects of RSV and of
other respiratory viruses on 17q21 gene-expression
levels would allow us to determine whether the
increased expression levels of ORMDL3 and GSDMB
are specific to HRV stimulation.
In summary, this study establishes a role of
17q21 variants in the development of HRV wheezing illnesses during early childhood and shows
that the effects of the 17q21 genotype on the
susceptibility to asthma are seen primarily in the
subgroup of children who have had an HRV wheezing illness in early childhood. Our results underscore the importance of studying the joint effects of genetic and environmental risk factors to
gain a fuller understanding of the mechanisms
underlying complex diseases.
Supported by grants (HL070831 and AI070503) from the National Institutes of Health. Dr. Jackson was supported by KL2
grant UL1TR000427 from the National Institutes of Health. The
Lundbeck Foundation, Danish Council for Strategic Research,
and Danish Pediatric Asthma Center provided the core funding
for the COPSAC research unit.
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
We thank Nick Levinsky for his help with the HRV stimulation protocols; Maitane Arruabarrena Orbegozo for technical
assistance; Dagan Loisel, Darren Cusanovich, and Yoav Gilad for
helpful discussions and comments on an earlier draft of the manuscript; all the study participants; and the COAST and COPSAC
research teams.
References
1. Moffatt MF, Kabesch M, Liang L, et al.
Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma. Nature 2007;448:470-3.
2. Akhabir L, Sandford AJ. Genomewide association studies for discovery of
genes involved in asthma. Respirology
2011;16:396-406.
3. Ober C, Yao TC. The genetics of asthma and allergic disease: a 21st century perspective. Immunol Rev 2011;242:10-30.
4. Bouzigon E, Corda E, Aschard H, et
al. Effect of 17q21 variants and smoking
exposure in early-onset asthma. N Engl J
Med 2008;359:1985-94.
5. Halapi E, Gudbjartsson DF, Jonsdottir
GM, et al. A sequence variant on 17q21 is
associated with age at onset and severity of
asthma. Eur J Hum Genet 2010;18:902-8.
6. Moffatt MF, Gut IG, Demenais F, et al.
A large-scale, consortium-based genome-
1406
wide association study of asthma. N Engl

J Med 2010;363:1211-21.
7. Bisgaard H, Bnnelykke K, Sleiman PM,
et al. Chromosome 17q21 gene variants
are associated with asthma and exacerbations but not atopy in early childhood. Am
J Respir Crit Care Med 2009;179:179-85.
8. Wu H, Romieu I, Sienra-Monge JJ, Li
H, del Rio-Navarro BE, London SJ. Genetic variation in ORM1-like 3 (ORMDL3)
and gasdermin-like (GSDML) and childhood asthma. Allergy 2009;64:629-35.
9. Flory JH, Sleiman PM, Christie JD, et
al. 17q12-21 Variants interact with smoke
exposure as a risk factor for pediatric
asthma but are equally associated with
early-onset versus late-onset asthma in
North Americans of European ancestry.
J Allergy Clin Immunol 2009;124:605-7.
10. Smit LA, Bouzigon E, Pin I, et al.
17q21 Variants modify the association be-
tween early respiratory infections and

asthma. Eur Respir J 2010;36:57-64.
11. van der Valk RJ, Duijts L, Kerkhof M,
et al. Interaction of a 17q12 variant with
both fetal and infant smoke exposure in
the development of childhood asthmalike symptoms. Allergy 2012;67:767-74.
12. Verlaan DJ, Ge B, Grundberg E, et al.
Targeted screening of cis-regulatory variation in human haplotypes. Genome Res
2009;19:118-27.
13. Murphy A, Chu JH, Xu M, et al. Mapping of numerous disease-associated expression polymorphisms in primary peripheral blood CD4+ lymphocytes. Hum
Mol Genet 2010;19:4745-57.
14. Ober C, Vercelli D. Gene-environment
interactions in human disease: nuisance
or opportunity? Trends Genet 2011;27:
107-15.
15. Sykes A, Johnston SL. Etiology of


asthma exacerbations. J Allergy Clin Immunol 2008;122:685-8.
16. Johnston SL, Pattemore PK, Sanderson G, et al. Community study of role of
viral infections in exacerbations of asthma in 9-11 year old children. BMJ 1995;
310:1225-9.
17. Bisgaard H, Hermansen MN, Bnne
lykke K, et al. Association of bacteria and
viruses with wheezy episodes in young
children: prospective birth cohort study.
BMJ 2010;341:c4978.
18. Kotaniemi-Syrjnen A, Vainionp R,
Reijonen TM, Waris M, Korhonen K, Korp
pi M. Rhinovirus-induced wheezing in infancy the first sign of childhood asthma?
19. Hyvrinen MK, Kotaniemi-Syrjnen A,
Reijonen TM, Korhonen K, Korppi MO.
Teenage asthma after severe early childhood wheezing: an 11-year prospective
follow-up. Pediatr Pulmonol 2005;40:31623.
20. Jackson DJ, Gangnon RE, Evans MD,
et al. Wheezing rhinovirus illnesses in
early life predict asthma development in
high-risk children. Am J Respir Crit Care

Med 2008;178:667-72.
21. Lemanske RF Jr. The Childhood Origins of Asthma (COAST) study. Pediatr
Allergy Immunol 2002;13:Suppl 15:38-43.
22. Bisgaard H, Pipper CB, Bnnelykke K.
Endotyping early childhood asthma by
quantitative symptom assessment. J Allergy Clin Immunol 2011;127(5):1155.e21164.e2.
23. DiFranza JR, Aligne CA, Weitzman M.
Prenatal and postnatal environmental tobacco smoke exposure and childrens
health. Pediatrics 2004;113:1007-15.
24. Hirota T, Harada M, Sakashita M, et
al. Genetic polymorphism regulating
ORM1-like 3 (Saccharomyces cerevisiae)
expression is associated with childhood
atopic asthma in a Japanese population.
25. Cantero-Recasens G, Fandos C, RubioMoscardo F, Valverde MA, Vicente R. The
asthma-associated ORMDL3 gene product
regulates endoplasmic reticulum-mediated
calcium signaling and cellular stress. Hum
Mol Genet 2010;19:111-21.
26. Miller M, Tam AB, Cho JY, et al. ORMDL3 is an inducible lung epithelial gene
regulating metalloproteases, chemokines,
OAS, and ATF6. Proc Natl Acad Sci U S A
2012;109:16648-53.
27. Trujillo-Alonso V, Maruri-Avidal L,
Arias CF, Lpez S. Rotavirus infection induces the unfolded protein response of
the cell and controls it through the nonstructural protein NSP3. J Virol 2011;85:
12594-604.
28. Carl-McGrath S, Schneider-Stock R,
Ebert M, Rocken C. Differential expression and localisation of gasdermin-like
(GSDML), a novel member of the cancerassociated GSDMDC protein family, in
neoplastic and non-neoplastic gastric, hepatic, and colon tissues. Pathology 2008;
40:13-24.
29. Saeki N, Usui T, Aoyagi K, et al. Distinctive expression and function of four
GSDM family genes (GSDMA-D) in normal and malignant upper gastrointestinal
epithelium. Genes Chromosomes Cancer
2009;48:261-71.
Copyright 2013 Massachusetts Medical Society.
journal archive at nejm.org
Every article published by the Journal is now available at NEJM.org, beginning

with the first article published in January 1812. The entire archive is fully searchable,
and browsing of titles and tables of contents is easy and available to all.
Individual subscribers are entitled to free 24-hour access to 50 archive articles per year.
Access to content in the archive is available on a per-article basis and is also
being provided through many institutional subscriptions.

1407

Rhinovirus Wheezing Illness and Genetic Risk of Childhood-Onset Asthma

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Rhinovirus Wheezing Illness and Genetic Risk of Childhood-Onset Asthma

Uploaded by

Copyright:

Available Formats

The

Rhinovirus Wheezing Illness and Genetic

This article was published on March 27,

N Engl J Med 2013;368:1398-407.

n engl j med 368;15 nejm.org april 11, 2013

The New England Journal of Medicine

Wheezing and Genetic Risk of Asthma

he first genomewide association

We included data from two cohorts of children

n engl j med 368;15 nejm.org april 11, 2013

The New England Journal of Medicine

illnesses in the COPSAC cohort are provided in the

Five asthma-associated 17q21 single-nucleotide

covariates. In addition, the number of wheezing

A blood sample (20 ml) was obtained from each

Complementary DNA (cDNA) was synthesized with

n engl j med 368;15 nejm.org april 11, 2013

The New England Journal of Medicine

Wheezing and Genetic Risk of Asthma

* HRV denotes human rhinovirus, and RSV respiratory syncytial virus.

All the SNPs showed modest associations with

and Fig. 1B) in the first 3 years of life. None of

We next examined interactions between the 17q21

n engl j med 368;15 nejm.org april 11, 2013

The New England Journal of Medicine

B Mean No. of HRV Wheezing Illnesses

C Prevalence of Asthma According to History of HRV

D Asthma Risk, Children with HRV Wheezing Illnesses

Odds Ratio (log10)

P=0.004 for interaction

E Asthma Risk, Children without HRV Wheezing Illnesses

Odds Ratio (log10)

No. with Genotype

n engl j med 368;15 nejm.org april 11, 2013

The New England Journal of Medicine

Wheezing and Genetic Risk of Asthma

A Prevalence of Asthma According to History of HRV

B Asthma Risk, Children with HRV Wheezing Illnesses

Odds Ratio (log10)

P=0.08 for interaction

C Asthma Risk, Children without HRV Wheezing Illnesses

Odds Ratio (log10)

Replication Studies in the COPSAC Cohort

We next studied this interaction in the COPSAC

hort than in the COAST cohort (24.6% vs. 31.3%).

n engl j med 368;15 nejm.org april 11, 2013

The New England Journal of Medicine

from 100 adults. Of the five genes at this locus,

n engl j med 368;15 nejm.org april 11, 2013

The New England Journal of Medicine

Wheezing and Genetic Risk of Asthma

Figure 4. Association between rs7216389 Genotype

We have found a significant interaction, in both

due to insufficient power. Second, we analyzed

n engl j med 368;15 nejm.org april 11, 2013

The New England Journal of Medicine

underlying predisposition for asthma and is not

epithelia.29 Further studies of the effects of the

wide association study of asthma. N Engl

tween early respiratory infections and

n engl j med 368;15 nejm.org april 11, 2013

The New England Journal of Medicine